Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0802—Tripeptides with the first amino acid being neutral
  • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/06—Tripeptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/2013—IL-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/204—IL-6
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/208—IL-12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
  • A61K38/212—IFN-alpha
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link

Definitions

• the present disclosure is generally directed to antiviral compounds, and more specifically directed to compounds which inhibit the function of the NS3 protease (also referred to herein as "serine protease") encoded by Hepatitis C virus (HCV), compositions comprising such compounds, and methods for inhibiting the function of the NS3 protease.
• NS3 protease also referred to herein as "serine protease”
• HCV Hepatitis C virus
• HCV is a major human pathogen, infecting an estimated 170 million persons worldwide - roughly five times the number infected by human immunodeficiency virus type 1. A substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cirrhosis and hepatocellular carcinoma.
• the first one cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N -terminal region of NS3 and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A- NS4B, NS4B-NS5A, NS5A-NS5B sites.
• the NS4A protein appears to serve multiple functions, acting as a co-factor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components.
• the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein:
• the present disclosure provides a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, 2, 3, 4, 5, or 6; each R 1 is independently selected from alkoxy, alkyl, carboxamido, carboxy, cyano, cycloalkyloxy, dialkylamino, halo, haloalkyl, haloalkoxy, and phenyl, wherein the phenyl is optionally substituted with one or two groups independently selected from alkoxy, alkyl, halo, haloalkoxy, and haloalkyl; R 2 is selected from hydrogen, alkyl, halo, and haloalkyl; and R 3 is selected from alkoxycarbonyl, alkylcarbonyl, haloalkoxycarbonyl, haloalkylcarbonyl, and phenylcarbonyl, wherein the phenyl is optionally substituted with one or two groups independently selected from alkyl and halo.
• the present disclosure provides a composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• a composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, at least one additional compound having anti-HCV activity, and a pharmaceutical carrier.
• at least one of the additional compounds is an interferon or a ribavirin.
• the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
• the present disclosure provides a compound as described above for use in a method of treating an HCV infection in a patient, said method comprising administering to the patient a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• the method further comprises administering at least one additional compound having anti-HCV activity prior to, after, or simultaneously with the compound of formula (I), or a pharmaceutically acceptable salt thereof.
• at least one of the additional compounds is an interferon or a ribavirin.
• the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
• the present disclosure provides the said compound for use in a method of treating an HCV infection in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and at least one additional compound having anti-HCV activity prior to, after, or simultaneously with the compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein at least one of the additional compounds is selected from interleukin 2, interleukin 6, interleukin 12, Imiquimod, ribavirin, an inosine 5'-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
• alkoxy refers to an alkyl group attached to the parent molecular moiety through an oxygen atom.
• alkoxyalkyl refers to an alkyl group substituted with one, two, or three alkoxy groups.
• alkoxycarbonyl refers to an alkoxy group attached to the parent molecular moiety through a carbonyl group.
• alkyl refers to a group derived from a straight or branched chain saturated hydrocarbon containing from one to ten carbon atoms.
• alkylcarbonyl refers to an alkyl group attached to the parent molecular moiety through a carbonyl group.
• alkylamino refers to -NHR q , wherein R q is alkyl.
• alkylsulfonyl refers to an alkyl group attached to the parent molecular moiety through a sulfonyl group.
• carbonyl refers to -C(O)-.
• cyano refers to -CN.
• cycloalkyl refers to a saturated monocyclic or bicyclic hydrocarbon ring system having three to seven carbon atoms and zero heteroatoms.
• Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, and cyclopentyl.
• cycloalkylalkoxy refers to a (cycloalkyl)alkyl group attached to the parent molecular moiety through an oxygen atom.
• cycloalkylalkoxycarbonyl refers to a cycloalkylalkoxy group attached to the parent molecular moiety through a carbonyl group.
• (cycloalkyl)alkyl refers to an alkyl group substituted with one, two, or three cycloalkyl groups.
• cycloalkylcarbonyl refers to a cycloalkyl group attached to the parent molecular moiety through a carbonyl group.
• cycloalkyloxy refers to a cycloalkyl group attached to the parent molecular moiety through an oxygen atom.
• cycloalkyloxycarbonyl refers to a cycloalkyloxy group attached to the parent molecular moiety through a carbonyl group.
• deuteroalkoxy refers to an alkoxy group wherein at least one of the hydrogen atoms is replaced by a deuterium atom.
• deuteroalkoxycarbonyl refers to a deuteroalkoxy group attached to the parent molecular moiety through a carbonyl group.
• deuterohaloalkoxy refers to a haloalkoxy group where at least one of the hydrogen atoms is replaced by a deuterium atom.
• deuterohaloalkoxycarbonyl refers to a deuterohaloalkoxy group attached to the parent molecular moiety through a carbonyl group.
• dialkylamino refers to -NR p R q , wherein R p and R q are alkyl groups.
• the alkyl groups may be the same or different.
• dialkylaminocarbonyl refers to a dialkylamino group attached to the parent molecular moiety through a carbonyl group.
• dialkylaminocarbonylcarbonyl refers to a dialkylaminocarbonyl group attached to the parent molecular moiety through a carbonyl group.
• halo and halogen, as used herein, refer to F, Cl, Br, and I.
• haloalkoxy refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.
• haloalkoxycarbonyl refers to a haloalkoxy group attached to the parent molecular moiety through a carbonyl group.
• haloalkyl refers to an alkyl group substituted with one, two, three, or four halogen atoms.
• haloalkylcarbonyl refers to a haloalkyl group attached to the parent molecular moiety through a carbonyl group.
• haloalkylamino refers to -NHR q , wherein R q is a haloalkyl group.
• haloalkylaminocarbonyl refers to a haloalkylamino group attached to the parent molecular moiety through a carbonyl group.
• haloalkylcarbonyl refers to a haloalkyl group attached to the parent molecular moiety through a carbonyl group.
• heterocyclyl refers to a five-, six-, or seven-membered ring containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• the five-membered ring has zero to two double bonds and the six- and seven-membered rings have zero to three double bonds.
• heterocyclyl also includes bicyclic groups in which the heterocyclyl ring is fused to a phenyl group, a monocyclic cycloalkenyl group, a monocyclic cycloalkyl group, or another monocyclic heterocyclyl group; and tricyclic groups in which a bicyclic system is fused to a phenyl group, a monocyclic cycloalkenyl group, a monocyclic cycloalkyl group, or another monocyclic heterocyclyl group.
• the heterocyclyl groups of the present invention can be attached to the parent molecular moiety through a carbon atom or a nitrogen atom in the group.
• heterocyclyl groups include, but are not limited to, benzothienyl, furyl, imidazolyl, indolinyl, indolyl, isothiazolyl, isoxazolyl, morpholinyl, oxazolyl, piperazinyl, piperidinyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrrolopyridinyl, pyrrolyl, thiazolyl, thienyl, and thiomorpholinyl.
• heterocyclylcarbonyl refers to a heterocyclyl group attached to the parent molecular moiety through a carbonyl group.
• heterocyclyloxy refers to a heterocyclyl group attached to the parent molecular moiety through an oxygen atom.
• heterocyclyloxycarbonyl refers to a heterocyclyloxy group attached to the parent molecular moiety through a carbonyl group.
• hydroxy refers to -OH.
• hydroxyalkyl refers to an alkyl group substituted with one, two, or three hydroxy groups.
• phenylcarbonyl refers to a phenyl group attached to the parent molecular moiety through a carbonyl group.
• phenyloxy refers to a phenyl group attached to the parent molecular moiety through an oxygen atom.
• phenyloxycarbonyl refers to a phenyloxy group attached to the parent molecular moiety through a carbonyl group.
• sulfonyl refers to -SO 2 -.
• the compounds of the present disclosure can exist as pharmaceutically acceptable salts.
• pharmaceutically acceptable salt represents salts or zwitterionic forms of the compounds of the present disclosure which are water or oil-soluble or dispersible, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
• the salts can be prepared during the final isolation and purification of the compounds or separately by reacting a suitable basic functionality with a suitable acid.
• Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate; digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbon
• Basic addition salts can be prepared during the final isolation and purification of the compounds by reacting an acidic group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
• a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
• the cations of pharmaceutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N- dimethylaniline, N- methylpiperidine, N- methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N- dibenzylphenethylamine, and N,N'- dibenzylethylenediamine.
• Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.
• anti-HCV activity means the compound is effective to treat the HCV virus.
• patient includes both human and other mammals.
• composition means a composition comprising a compound of the disclosure in combination with at least one additional pharmaceutical carrier, i.e., adjuvant, excipient or vehicle, such as diluents, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms.
• additional pharmaceutical carrier i.e., adjuvant, excipient or vehicle, such as diluents, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms.
• additional pharmaceutical carrier i.e., adjuvant, excipient
• phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable risk/benefit ratio.
• terapéuticaally effective amount means the total amount of each active component that is sufficient to show a meaningful patient benefit, e.g., a sustained reduction in viral load.
• a meaningful patient benefit e.g., a sustained reduction in viral load.
• the term refers to that ingredient alone.
• the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
• treat and “treating” refers to: (i) preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and/or (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition.
• the designations P1', P1, P2, P2*, P3, and P4, as used herein, map the relative positions of the amino acid residues of a protease inhibitor binding relative to the binding of the natural peptide cleavage substrate. Cleavage occurs in the natural substrate between P1 and P1' where the nonprime positions designate amino acids starting from the C-terminus end of the peptide natural cleavage site extending towards the N -terminus; whereas, the prime positions emanate from the N -terminus end of the cleavage site designation and extend toward the C-terminus.
• P1' refers to the first position away from the right hand end of the C-terminus of the cleavage site (i.e. N -terminus first position); whereas P1 starts the numbering from the left hand side of the C-terminus cleavage site, P2: second position from the C-terminus, etc.).
• the compounds may include P1 cyclopropyl element of formula wherein C 1 and C 2 each represent an asymmetric carbon atom at positions 1 and 2 of the cyclopropyl ring. It should be understood that the disclosure encompasses all stereochemical forms, or mixtures thereof, which possess the ability to inhibit HCV protease.
• Certain compounds of the present disclosure may also exist in different stable conformational forms which may be separable. Torsional asymmetry due to restricted rotation about an asymmetric single bond, for example because of steric hindrance or ring strain, may permit separation of different conformers.
• the present disclosure includes each conformational isomer of these compounds and mixtures thereof.
• Certain compounds of the present disclosure may exist in zwitterionic form and the present disclosure includes each zwitterionic form of these compounds and mixtures thereof.
• compositions which include therapeutically effective amounts of compounds of formula (I) or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
• the compounds of formula (I) and pharmaceutically acceptable salts thereof are as described above.
• the carrier(s), diluent(s), or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• a process for the preparation of a pharmaceutical formulation including admixing a compound of formula (I), or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.
• compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Dosage levels of between about 0.01 and about 150 milligram per kilogram (“mg/kg”) body weight per day, preferably between about 0.05 and about 100 mg/kg body weight per day of the compounds of the disclosure are typical in a monotherapy for the prevention and treatment of HCV mediated disease. Typically, the pharmaceutical compositions of this disclosure will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
• mg/kg milligram per kilogram
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending on the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the compound employed, the duration of treatment, and the age, gender, weight, and condition of the patient.
• Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
• treatment is initiated with small dosages substantially less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
• the compound is most desirably administered at a concentration level that will generally afford antivirally effective results without causing any harmful or deleterious side effects.
• compositions of this disclosure comprise a combination of a compound of the disclosure and one or more additional therapeutic and/or prophylactic agent
• both the compound and the additional agent can be present in a dose that is less than or equal to the dosage normally administered in a monotherapy regimen.
• the compositions of this disclosure may be co-formulated with one or more additional therapeutic or prophylactic agents, for example, in the form of a monolithic and/or bi/multi-layer tablet or may be administered separately from the therapeutic or prophylactic agent(s).
• compositions may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intracutaneous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous, or intradermal injections or infusions) route.
• Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
• compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil emulsions.
• the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
• an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
• Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agent can also be present.
• Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin sheaths.
• Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol can be added to the powder mixture before the filling operation.
• a disintegrating or solubilizing agent such as agar-agar, calcium carbonate, or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.
• suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, and the like.
• Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like.
• Disintegrators include, without limitation, starch, methyl cellulose, agar, betonite, xanthan gum, and the like.
• Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets.
• a powder mixture is prepared by mixing the compound, suitable comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelating, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or and absorption agent such as betonite, kaolin, or dicalcium phosphate.
• a binder such as carboxymethylcellulose, an aliginate, gelating, or polyvinyl pyrrolidone
• a solution retardant such as paraffin
• a resorption accelerator such as a quaternary salt and/or
• absorption agent such as betonite, kaolin, or dicalcium phosphate.
• the powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen.
• a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen.
• the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules.
• the granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc, or mineral oil.
• the lubricated mixture is then compressed into tablets.
• the compounds of the present disclosure can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps.
• a clear or opaque protective coating consisting of a sealing coat of shellac,
• Oral fluids such as solution, syrups, and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound.
• Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic vehicle.
• Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners, and the like can also be added.
• dosage unit formulations for oral administration can be microencapsulated.
• the formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax, or the like.
• the compounds of formula (I), and pharmaceutically acceptable salts thereof, can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
• liposomes can be formed from a variety of phopholipids, such as cholesterol, stearylamine, or phophatidylcholines.
• the compounds of formula (I) and pharmaceutically acceptable salts thereof may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
• the compounds may also be coupled with soluble polymers as targetable drug carriers.
• Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palitoyl residues.
• the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.
• a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.
• compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
• the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986 ).
• compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
• the formulations are preferably applied as a topical ointment or cream.
• the active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
• the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in oil base.
• compositions adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
• compositions adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.
• compositions adapted for rectal administration may be presented as suppositories or as enemas.
• compositions adapted for nasal administration wherein the carrier is a solid include a course powder which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
• Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or nasal drops, include aqueous or oil solutions of the active ingredient.
• Fine particle dusts or mists which may be generated by means of various types of metered, dose pressurized aerosols, nebulizers, or insufflators.
• compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.
• compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and soutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
• formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
• Table 1 below lists some illustrative examples of compounds that can be administered with the compounds of this disclosure.
• the compounds of the disclosure can be administered with other anti-HCV activity compounds in combination therapy, either jointly or separately, or by combining the compounds into a composition.
• Table 1 Brand Name Physiological Class Type of Inhibitor or Target Source Company NIM811 Cyclophilin Inhibitor Novartis Zadaxin Immuno-modulator Sciclone Suvus Methylene blue Bioenvision Actilon (CPG10101) TLR9 agonist Coley Batabulin (T67) Anticancer ⁇ -tubulin inhibitor Tularik Inc., South San Francisco, CA ISIS 14803 Antiviral antisense ISIS Phamaceuticals Inc, Carlsbad, CA/Elan Phamaceuticals Inc., New York, NY Summetrel Antiviral antiviral Endo Pharmaceuticals Holdings Inc., Chadds Ford, PA Brand Nane Physiological Class Type of Inhibitor or Target Source Company GS-9132 (ACH-
• the compounds of the disclosure may also be used as laboratory reagents.
• Compounds may be instrumental in providing research tools for designing of viral replication assays, validation of animal assay systems and structural biology studies to further enhance knowledge of the HCV disease mechanisms. Further, the compounds of the present disclosure are useful in establishing or determining the binding site of other antiviral compounds, for example, by competitive inhibition.
• the compounds of this disclosure may also be used to treat or prevent viral contamination of materials and therefore reduce the risk of viral infection of laboratory or medical personnel or patients who come in contact with such materials, e.g., blood, tissue, surgical instruments and garments, laboratory instruments and garments, and blood collection or transfusion apparatuses and materials.
• materials e.g., blood, tissue, surgical instruments and garments, laboratory instruments and garments, and blood collection or transfusion apparatuses and materials.
• This disclosure is intended to encompass compounds having formula (I) when prepared by synthetic processes or by metabolic processes including those occurring in the human or animal body (in vivo ) or processes occurring in vitro.
• reaction mass was allowed to come to room temperature and stirred overnight.
• the reaction mass was quenched with saturated ammonium chloride solution and extracted with diethyl ether (200 mL x 3).
• the combined organic layer was dried over anhydrous Na 2 SO 4 and concentrated at room temperature to get crude compound.
• the crude compound was purified by column chromotography (Silica gel, 10% TBME in pet ether) to get (2R)-6-methyloct-7-en-2-ol (12.4 g, 92%) as oily liquid.
• Step 1 Preparation of (2S,4R)-methyl 1-((2S,3R)-2-(tert-butoxycarbonylamino)-3,7-dimethylnon-8-enoyl)-4-hydroxypyrrolidine-2-carboxylate
• Step 3 Preparation of (2S,4R)-1-((2S,3R)-2-(tert-butoxycarbonylamino)-3,7-dimethylnon - 8 - enoyl) - 4 -( 4 - methoxyisoquinolin - 1 - yloxy)pyrrolidine - 2 - carboxylic acid
• Compound 2 tert-butyl (2R,6S,7R,11S,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(4-methoxyisoquinolin-1-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Step 1 Preparation of tert-butyl (2S,3R)-1-((2S,4R)-2-((1R,2S)-1-cyclopropylsulfonylcarbamoyl)-2 - vinylcyclopropylcarbamoyl)-4-hydroxypyrrolidin-1-yl)-3,7-dimethyl-1-oxonon-8-en-2-ylcarbamate
• Step 2 Preparation of tert-butyl tert-butyl (2R,6S,7R,13aS,14aR,16aS,Z)-14a- ( cyclopropylsulfonylcarbamoyl )- 2 - hydroxy - 7 , 11 - dimethyl - 5 , 16 - dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[ e ]pyrrolo[1 , 2 - a][1 , 4]diazacyclopentadecin - 6-ylcarbamate
• Step 3 Preparation of tert-butyl (2R, 6S, 7R, 13aS, 14aR, 16aS,Z)-2-(5-chloro-4-methoxyisoquinolin - 1 - yloxy )- 14a -( cyclopropylsulfonylcarbamoyl )- 7 , 11 - dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate
• Compound 8 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11S,13aS,14aR,16aS,Z)-2-(5-chloro-4-methoxyisoquinolin-1-yloxy)-14a-(cyclopropylsulfonylcarbamoyl)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• MS MS: MS m / z 843.3 (M + +1).
• Compound 9 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(6-methoxyisoquinolin-1-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Step 2 Preparation of 5 - choroisoquinolin - 1 ( 2H )- one
• Step 4 Preparation of 1 , 5 - dichloro - 4 - methoxyisoquinoline
• Step 1 Preparation of 7 - fluoro - 4 , 6 - dimethoxyisoquinolin - 1 ( 2H )- one
• Step 2 Preparation of 1 - chloro - 7 - fluoro - 4 , 6 - dimethoxyisoquinoline
• Step 3 Preparation of 1 , 7 - difluoro - 4 , 6 - dimethoxyisoquinoline
• Step 1 Preparation of ( E )- 3 -( 3 - methoxyphenyl ) acrylic azide
• Step 3 Preparation of 7 - fluoro - 4 - methoxyisoquinolin-1(2H)-one
• Step 4 Preparation of l - chloro - 7 - fluoro - 4 - methoxyisoquinoline
• Step 1 Preparation of 6 - methoxylquinoxalin - 2(1H)-one and 7-methoxyquinoxalin-2(1H)-one
• Steps 2 and 3 Preparation of 2-chloro-6-methoxyquinoxaline and 3 - chloro - 6-methoxyquinoxaline
• Step 1 Preparation of 1-chloro - 4 - ethoxyisoquinoline
• Step 1 Preparation of tert-butyl cyclopropylsulfonylcarbamate
• Step 2 Preparation of tert-butyl (1-methylcyclopropyl) sulfonylcarbamate
• Step 1 Preparation of tert-butyl 1-(hydroxymethyl)cyclopropylsulfonylcarbamate
• tert-butyl cyclopropylsulfonylcarbamate (30 g, 136 mmol) in 750 mL of THF was added dropwise butyllithium (1.6 M in hexane, 212 mL, 339 mmol) over 30 min at -78 °C and the resulting mixture was stirred at -78 °C for 1 h.
• Formaldehyde gas was generated from para-formaldehyde (by heating at 180 °C) and was purged into the above reaction mass for 30 min at -30 °C. The reaction was stirred at the same temperature for 1 h, then allowed to warm to room temperature.
• Step 2 Preparation of tert-butyl (1-(fluoromethyl)cyclopropyl)sulfonylcabamate
• Compound 11 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 5.
• Compound 11 tert-butyl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-2-(4,6-dimethoxyisoquinolin-1-yloxy)-14a-(1-(fluoromethyl)cyclopropylsulfonylcarbamoyl)-7,11 -dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 13 1,1,1-trifluoro-2-methylpropan-2-yl ((2R,6S,7R,11S,13aS,14aR,16aS,Z)-2-((4,6-dimethoxyisoquinolin-1-yl)oxy)-14a-(((1-(fluoromethyl)cyclopropyl)sulfonyl)carbamoyl)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate.
• Compound 14 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 7.
• Compound 14 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS, 14aR,16aS,Z)-2-(4,6-dimethoxyisoquinolin-1-yloxy)-7,11-dimethyl-14a-(1-methylcyclopropylsulfonylcarbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 15 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-2-(7-fluoro-4,6-dimethoxyisoquinolin-1-yloxy)-7,11-dimethyl-14a-(1-methylcyclopropylsulfonylcarbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 16 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11S,13aS,14aR,16aS,Z)-2-(7-fluoro-4,6-dimethoxyisoquinolin-1-yloxy)-7,11-dimethyl-14a-(1-methylcyclopropylsulfonylcarbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 18 1,1,1-trifluoro-2-methylpropan-2-yl ((2R,6S,7R,11S,13aS,14aR,16aS,Z)-2-((7-methoxyquinoxalin-2-yl)oxy)-7,11-dimethyl-14a-(((1-methylcyclopropyl)sulfonyl)carbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate.
• Compound 19 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR, 16aS,Z)-2-(4-ethoxyisoquinolin-1-yloxy)-7,11-dimethyl-14a-(1-methylcyclopropylsulfonylcarbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 20 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11S,13aS,14aR, 16aS,Z)-2-(4-ethoxyisoquinolin-1-yloxy)-7,11-diethyl-14a-(1-methylcyclopropylsulfonylcarbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 21 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 5.
• Compound 21 tert-butyl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(4,6-dimethoxyisoquinolin-1-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 22 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 7.
• Compound 22 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS, 14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(4,6-dimethoxyisoquinolin-1-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 23 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 5.
• Compound 23 tert-butyl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(7-fluoro-4-methoxyisoquinolin-1-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a] [1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 24 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 7.
• Compound 24 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(7-fluoro-4-methoxyisoquinolin-1-yloxy)-7,11 -dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 25 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 7.
• Compound 25 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-2-(5-chloro-4-methoxyisoquinolin-1-yloxy)-7,11-dimethyl-14a-(1-methylcyclopropylsulfonylcarbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 26 tert-butyl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(6-methoxyquinoxalin-2-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 28 tert-butyl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(7-methoxyquinoxalin-2-yloxy)-7,11 -dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 30 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(7-methoxyquinoxalin-2-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 31 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11S,13aS, 14aR, 16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(7-methoxyquinoxalin-2-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 32 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 7.
• Compound 32 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(6-methoxyquinoxalin-2-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 33 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 7.
• Compound 33 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(7-fluoro-4,6-dimethoxyisoquinolin-1-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 34 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 7.
• Compound 34 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS,14aR,16aS,Z)-14a-(cyclopropylsulfonylcarbamoyl)-2-(isoquinolin-1-yloxy)-7,11-dimethyl-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• Compound 35 was prepared using the intermediates described herein and by following the general procedure described for the synthesis of Compound 7.
• Compound 35 1,1,1-trifluoro-2-methylpropan-2-yl (2R,6S,7R,11R,13aS, 14aR, 16aS,Z)-2-(4-methoxyisoquinolin-1-yloxy)-7,11-dimethyl-14a-(1-methylcyclopropylsulfonylcarbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-ylcarbamate.
• HCV NS3/4A protease complex enzyme assays and cell-based HCV replicon assays were utilized in the present disclosure, and were prepared, conducted and validated as follows:
• HCV NS3 protease complexes derived from the BMS strain, H77 strain or J4L6S strain, were generated, as described below. These purified recombinant proteins were generated for use in a homogeneous assay (see below) to provide an indication of how effective compounds of the present disclosure would be in inhibiting HCV NS3 proteolytic activity.
• Serum from an HCV-infected patient was obtained from Dr. T. Wright, San Francisco Hospital.
• An engineered full-length cDNA (compliment deoxyribonucleic acid) template of the HCV genome was constructed from DNA fragments obtained by reverse transcription-PCR (RT-PCR) of serum RNA (ribonucleic acid) and using primers selected on the basis of homology between other genotype la strains. From the determination of the entire genome sequence, a genotype la was assigned to the HCV isolate according to the classification of Simmonds et al. (See P Simmonds, KA Rose, S Graham, SW Chan, F McOmish, BC Dow, EA Follett, PL Yap and H Marsden, J. Clin.
• the amino acid sequence of the nonstructural region, NS2-5B was shown to be >97% identical to HCV genotype 1a (H77) and 87% identical to genotype 1b (J4L6S).
• the infectious clones, H77 (1a genotype) and J4L6S (1b genotype) were obtained from R. Purcell (NIH) and the sequences are published in Genbank (AAB67036, see Yanagi,M., Purcell,R.H., Emerson,S.U. and Bukh, J. Proc. Natl. Acad. Sci. U.S.A.
• the H77 and J4L6S strains were used for production of recombinant NS3/4A protease complexes.
• DNA encoding the recombinant HCV NS3/4A protease complex (amino acids 1027 to 1711) for these strains was manipulated as described by P. Gallinari et al. (see Gallinari P, Paolini C, Brennan D, Nardi C, Steinkuhler C, De Francesco R. Biochemistry. 38(17):5620-32, (1999 )). Briefly, a three-lysine solubilizing tail was added at the 3'-end of the NS4A coding region.
• the cysteine in the P1 position of the NS4A-NS4B cleavage site (amino acid 1711) was changed to a glycine to avoid the proteolytic cleavage of the lysine tag. Furthermore, a cysteine to serine mutation was introduced by PCR at amino acid position 1454 to prevent the autolytic cleavage in the NS3 helicase domain.
• the variant DNA fragment was cloned in the pET21b bacterial expression vector (Novagen) and the NS3/4A complex was expressed in Escherichia. coli strain BL21 (DE3) (Invitrogen) following the protocol described by P. Gallinari et al.
• the cells were resuspended in lysis buffer (10 mL/g) consisting of 25 mM N -(2-Hydroxyethyl)Piperazine- N' -(2-Ethane Sulfonic acid) (HEPES), pH 7.5, 20% glycerol, 500 mM Sodium Chloride (NaCl), 0.5% Triton X-100, 1 microgram/milliliter (“ ⁇ g/mL”) lysozyme, 5 mM Magnesium Chloride (MgCl 2 ), 1 ⁇ g/ml DnaseI, 5 mM ⁇ -Mercaptoethanol ( ⁇ ME), Protease inhibitor-Ethylenediamine Tetraacetic acid (EDTA) free (Roche), homogenized and incubated for 20 minutes (min) at 4°C.
• lysis buffer 10 mL/g
• HEPES N -(2-Hydroxyethyl)Piperazin
• the homogenate was sonicated and clarified by ultra-centrifugation at 235000 g for 1 hour (h) at 4°C. Imidazole was added to the supernatant to a final concentration of 15 mM and the pH adjusted to 8.0.
• the crude protein extract was loaded on a Nickel-Nitrilotriacetic acid (Ni-NTA) column pre-equilibrated with buffer B (25 mM HEPES, pH 8.0, 20% glycerol, 500 mM NaCl, 0.5% Triton X-100, 15 mM imidazole, 5 mM ⁇ ME). The sample was loaded at a flow rate of 1 mL/min.
• NS3/4A protease complex-containing fractions were pooled and loaded on a desalting column Superdex-S200 pre-equilibrated with buffer D (25 mM HEPES, pH 7.5, 20% glycerol, 300 mM NaCl, 0.2% Triton X-100, 10 mM ⁇ ME). Sample was loaded at a flow rate of 1 mL/min. NS3/4A protease complex-containing fractions were pooled and concentrated to approximately 0.5 mg/ml. The purity of the NS3/4A protease complexes, derived from the BMS, H77 and J4L6S strains, were judged to be greater than 90% by SDS-PAGE and mass spectrometry analyses. The enzyme was stored at -80 °C, thawed on ice and diluted prior to use in assay buffer.
• This in vitro assay was to measure the inhibition of HCV NS3 protease complexes, derived from the BMS strain, H77 strain or J4L6S strain, as described above, by compounds of the present disclosure. This assay provides an indication of how effective compounds of the present disclosure would be in inhibiting HCV NS3 proteolytic activity.
• an NS3/4A peptide substrate was used.
• the substrate was RET S 1 (Resonance Energy Transfer Depsipeptide Substrate; AnaSpec, Inc. cat # 22991)(FRET peptide), described by Taliani et al. in Anal. Biochem. 240(2):60-67 (1996 ).
• the sequence of this peptide is loosely based on the NS4A/NS4B natural cleavage site for the HCV NS3 protease except there is an ester linkage rather than an amide bond at the cleavage site.
• the peptide also contains a fluorescence donor, EDANS, near one end of the peptide and an acceptor, DABCYL, near the other end.
• EDANS fluorescence donor
• DABCYL acceptor
• the fluorescence of the peptide is quenched by intermolecular resonance energy transfer (RET) between the donor and the acceptor, but as the NS3 protease cleaves the peptide the products are released from RET quenching and the fluorescence of the donor becomes apparent.
• RET intermolecular resonance energy transfer
• the peptide substrate was incubated with one of the three recombinant NS3/4A protease complexes, in the absence or presence of a compound of the present disclosure.
• the inhibitory effects of a compound were determined by monitoring the formation of fluorescent reaction product in real time using a Cytofluor Series 4000.
• HEPES and Glycerol were obtained from GIBCO-BRL.
• Dimethyl Sulfoxide (DMSO) was obtained from Sigma.
• ⁇ -Mercaptoethanol was obtained from Bio Rad.
• Assay buffer 50 mM HEPES, pH 7.5; 0.15 M NaCl; 0.1% Triton; 15% Glycerol; 10 mM ⁇ ME.
• Substrate 2 ⁇ M final concentration (from a 2 mM stock solution in DMSO stored at -20°C).
• HCV NS3/4A protease type 1a (1b) 2-3 nM final concentration (from a 5 ⁇ M stock solution in 25 mM HEPES, pH 7.5, 20% glycerol, 300 mM NaCl, 0.2% Triton-X100, 10 mM ⁇ ME).
• the assay was made more sensitive by adding 50 ⁇ g/ml Bovine Serum Albumin (Sigma) to the assay buffer and reducing the end protease concentration to 300 pM.
• the assay was performed in a 96-well polystyrene black plate from Falcon. Each well contained 25 ⁇ l NS3/4A protease complex in assay buffer, 50 ⁇ l of a compound of the present disclosure in 10% DMSO/assay buffer and 25 ⁇ l substrate in assay buffer. A control (no compound) was also prepared on the same assay plate. The enzyme complex was mixed with compound or control solution for 1 min before initiating the enzymatic reaction by the addition of substrate. The assay plate was read immediately using the Cytofluor Series 4000 (Perspective Biosystems). The instrument was set to read an emission of 340 nm and excitation of 490 nm at 25°C. Reactions were generally followed for approximately 15 min.
• the percent inhibition was calculated with the following equation: 100 ⁇ ⁇ F inh / ⁇ F con ⁇ 100 where ⁇ F is the change in fluorescence over the linear range of the curve.
• CDNA encoding a humanized form of the Renilla luciferase gene and a linker sequence fused directly to the 3'-end of the luciferase gene were introduced into the replicon construct using an Asc1 restriction site located in core, directly upstream of the neomycin marker gene.
• the adaptive mutation at position 1179 was also introduced ( Blight KJ, Kolykhalov, AA, Rice, CM, Science 290(5498): 1972-1974 ).
• a stable cell line constitutively expressing this HCV replicon construct was generated by first linearizing plasmid DNAs with ScaI.
• RNA transcripts were synthesized in vitro using the T7 MegaScript transcription kit (Ambion, Austin, TX) according to manufacturer's directions. In vitro transcripts of the cDNA were transfected into the human hepatoma cell line, HUH-7. Selection for cells constitutively expressing the HCV replicon was achieved in the presence of the selectable marker, neomycin (G418). Resulting cell lines were characterized for positive and negative strand RNA production and protein production over time.
• a stable HCV replicon luciferase reporter cell line representing the genotype 1a H77 strain ( Yanagi M, Purcell RH, Emerson SU, et al. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA 1997; 94(16):8738-8743 ) was generated as described previously for the genotype 1b(Con1) replicon luciferase cell line.
• the replicon construct was modified by introducing mutations were introduced into the genes encoding the NS3 helicase domain (proline replaced by leucine at position 1496) and NS5A (serine to isoleucine at position 2204) to improve replication in cell culture.
• HCV replicon luciferase assays were developed to monitor the inhibitory effects of compounds described in the disclosure on HCV genotypes 1a and 1b viral replication.
• HUH-7 cells constitutively expressing the HCV replicon, were grown in Dulbecco's Modified Eagle Media (DMEM) (Gibco-BRL) containing 10% Fetal calf serum (FCS) (Sigma) and 1 mg/mL G418 (Gibco-BRL).
• DMEM Dulbecco's Modified Eagle Media
• FCS Fetal calf serum
• G418 G418
• the plates were then seeded with 50 ⁇ L of cells at a density of 3.0 x 10 3 cells/well in DMEM containing 4 % FCS (final DMSO concentration at 0.5 %). After 3 days incubation at 37°C, cells were analyzed for Renilla Luciferase activity using the EnduRen as substrate (Promega cat #E6485). The EnduRen substrate was diluted in DMEM and then added to the plates to a final concentration of 7.5 ⁇ M. The plates were incubated for 2 hrs at 37°C and then read immediately for 30 seconds with Viewlux Imager (PerkinElmer) using a luminescence program.
• CC 50 values were generated by multiplexing the EnduRen-containing plates with Cell Titer-Blue (Promega, cat # G8082). Cell-Titer Blue (3 ⁇ L) was added to each well and incubated for 8 hrs at 37°C. The fluorescence signal from each well was read, with an excitation wavelength at 525/10 nm and an emission wavelength of 598/10 nm, using the Viewlux Imager.

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